C. elegans Knock-in Services
From IN Vivo Models - C. Elegans Solutions
The nematode Caenorhabditis elegans is widely used in genetic and biomedical research. DNA fragment insertions can be introduced into the C. elegans genome. Our C. elegans knock-in services include point mutations, floxed alleles, degron tagging, fluorescent tagging and immunotagging. In the past 5 years, InVivo Biosystems has made over 600 fluorescent transgenic lines includuing 15 different fluorophores, 8 of which are variants of GFP. Fluorescent Tagging. Intestinal nuclei from late L4 worms in an lmn-1p::GFP::LMN-1 transgenic strain. The image is lmn-1 tagged endogenously with GFP. Image courtesy of Dr. André Catic, Baylor College of Medicine.
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C. elegans Knock-in Service Details
Our Point Mutation service uses CRISPR/Cas9, which is the best gene editing method for creating small, precise edits to introduce a small number of nucleotide changes at a target site.
With this service you can:
- study a disease-causing mutation
- humanize a critical amino acid
- explore the binding site of an enzyme
- introduce phosphomimetics
- mutate isoform start sites or make any specific mutation of interest.
Among the F1 candidates with the Co-CRISPR edit, our average percentage of animals with the target edit was 65.7%.
Edit percentages as high as 95% were observed.
For these projects, our average time from injection to sequence confirmation of two independent homozygous lines was 15.3 days.
Our quickest project only took 10 days!
All 96 mutations were created in the STXBP1 gene (which is associated with epilepsy in humans) via CRISPR.
The worm homolog of STXBP1, unc-18, causes uncoordination and near-complete lack of pharyngeal pumping when knocked out.
The functionality is restored by replacing the worm gene with the coding sequence for human STXBP1.
Fluorescent tagging of genes is widely used in many model organisms to study which tissues a gene of interest is expressed in, where in the cell a gene is expressed, or to determine whether multiple genes of interest are expressed in the same location. C. elegans is particularly well suited to fluorescence studies because they are transparent. Fluorescence can be easily observed in both live and fixed images.
Knock-in a fluorescent protein at the endogenous locus using CRISPR gene editing technology.
Adding a fluorescent protein tag at the endogenous locus enables you to:
- detect your protein in-vivo at native gene expression levels
- ensure that you have the correct expression pattern
- identify protein-protein interactions
- confirm degradation of your protein when paired with a degron sequence
- visualize protein synthesis rates
- quantify protein levels
- visualize protein localization to organelles or membranes
You can choose from a variety of fluorophores including YFP, BFP, eGFP, mCherry, mOrange, mScarlet, and many more. All of our fluorescent fluorophores are optimized for expression in C. elegans via codon optimization and insertion of introns.
Use our fluorescent fluorophore tags in combination with the Fluorescent Protein RNAi to reduce or eliminate fluorophore expression.
Concerned that the addition of a fluorescent fluorophores will inhibit protein function? We can use a 2A or SL2 sequence to separate the 2 components!
Things to consider when ordering your strain:
- Fluorescent protein best suited for your experiments
- Best terminus to tag
We are not limited to a fixed list of fluorophores. If you have a fluor that you would like to use, you can provide us the sequence and we will incorporate it into the design of your transgenic project. If you need your fluorescent transgenics lines to be imaged, we can readily help you with that too! If you are unsure, our genetic engineering experts will be glad to advise you. Contact us.
For immobilization of live animals to image yourself, learn more about NemaGel.
The addition of an immunotag at the endogenous locus enables you to quantify your protein without altering the level of gene expression.
Immunotags are added using CRISPR/Cas9 gene editing technology.
You can choose from a variety of tags including FLAG, HA, HIS, TAP, or S-peptide.
All tags have been optimized for expression in C. elegans.
C. elegans Degron Tagging Service
You can knock-in a degron tag on an endogenous protein or fluorescent protein with our degron tagging service.
Adding a degron tag enables you to:
- use protein degradation as an complementary method to genetic knockouts or RNAi
- tag your protein of interested for degradation at the protein level
- control the timing of protein degradation with an inducer (e.g. auxin, blue light)
- confirm degradation of your protein when paired with a fluorescent sequence
- How do I get rid of This Pesky Protein? The Hows and Whys of Protein Degradation
You can choose from a variety of degron inducible and developmental systems including Auxin Inducible Degradation, Photosensitive or blue-light inducible degradation, degron-tagged reporters of membrane topology, and OMA-1 tags that work to degrade protein from the 1-cell stage.
Use our degron tags in combination with the fluorescent tagging to visualize protein expression and degradation.
Things to consider when ordering your strain:
- Degron tag best suited for your experiments
- Best terminus to tag
For auxin-inducible degradation, TIR1 is necessary to generate a functional recognition complex and achieve successful target degradation. If you do not have a TIR1-expressing strain ready, we can suggest the most suitable commercially available strain, or we can generate a custom TIR1 strain for you.
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